Anandamide modulates WNT-5A/BCL-2, IP3/NFATc1, and HMGB1/NF-κB trajectories to protect against mercuric chloride-induced acute kidney injury

Endocannabinoid anandamide (AEA) has a physiological role in regulating renal blood flow, whereas its analogs ameliorated renal ischemia/reperfusion injury. Nonetheless, the role of AEA against mercuric chloride (HgCl2)-induced renal toxicity has not been unraveled. Rats were allocated into control, HgCl2, and HgCl2/AEA treated groups. The administration of AEA quelled the HgCl2-mediated increase in inositol trisphosphate (IP3) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). The endocannabinoid also signified its anti-inflammatory potential by turning off the inflammatory cascade evidenced by the suppression of high mobility group box protein-1 (HMGB1), receptor of glycated end products (RAGE), nuclear factor-κB p65 (NF-κB), and unexpectedly PPAR-γ. Additionally, the aptitude of AEA to inhibit malondialdehyde and boost glutathione points to its antioxidant capacity. Moreover, AEA by enhancing the depleted renal WNT-5A and reducing cystatin-C and KIM-1 (two kidney function parameters) partly verified its anti-apoptotic capacity, confirmed by inhibiting caspase-3 and increasing B-cell lymphoma-2 (BCL-2). The beneficial effect of AEA was mirrored by the improved architecture and kidney function evidenced by the reduction in cystatin-C, KIM-1, creatinine, BUN, and caspase1-induced activated IL-18. In conclusion, our results verify the reno-protective potential of AEA against HgCl2-induced kidney injury by its anti-inflammatory, antioxidant, and anti-apoptotic capacities by modulating WNT-5A/BCL-2, IP3/NFATC1, HMGB-1/RAGE/NF-κB, caspase-1/IL-18, and caspase-3/BCL-2 cues.


Material and methods
Animals. week-old male Wistar rats (180-220 g) were purchased from the breeding colony at the animal house of the Research Institute of Medical Entomology (RIME, Giza, Egypt). Rats were allowed to acclimatize for a week before experiment was carried out; and they were housed under constant environmental conditions ( Induction of acute nephrotoxicity by HgCl 2 . Nephrotoxicity was induced by a single subcutaneous dose of HgCl 2 (3 mg/kg; Sigma-Aldrich, MO, USA) dissolved in saline 39 .
Experimental design. Rats were randomly divided into 3 groups (n = 6 each); in the first group, animals served as the control group, whereas those in the second group received HgCl 2 and were designated as the nephrotoxic group. Rats in the third group were injected with AEA (2.5 mg/kg; i.p; Tocris Bioscience, Bristol, UK) 30 min before and 24 h after the induction of AKI 40 (AEA group). Twenty-four hours after the last dose of AEA, animals were anesthetized by a high dose of thiopental (100 mg/kg) and blood was rapidly collected from the femoral vein to prepare sera samples to assess renal function. Afterward, the left kidney of all rats was excised, decapsulated, and homogenized in phosphate buffer saline (PBS) for biochemical assessments.
Homogenates were aliquoted for the determination of the intended parameters and stored at − 80 °C. The right kidney of 3 representative rats/group was used for histopathological analysis.
Biochemical analysis. Assessment of serum parameters. Serum creatinine was quantified by a purchased spectrophotometric assay kit (Bioassay Systems, CA, USA; cat# DICT-500) using an improved Jaffe method that utilizes picrate to form a red-colored complex with creatinine. Blood urea nitrogen (BUN) was evaluated spectrophotometrically using a colorimetric assay kit (Bioassay Systems; cat# DIUR-500) that depends on the modified Berthelot reaction. Serum levels of cystatin C (CUSABIO, MD, USA; cat# CSB-E08385r), kidney injury molecule-1 (KIM-1; MyBioSource, CA, USA; cat# MBS2702467), and IL-18 (LSBio, WA, USA; cat# LS-F2596) were determined using the corresponding rat ELISA kits. All experiments were processed according to the manufacturers' instructions.
Determination of renal caspase-3 activity. The activity of caspase-3 was determined using the colorimetric assay kit (Sigma-Aldrich; cat# CASP3C-1KT) that is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3. This results in the release of the p-nitroaniline (pNA) moiety that has a high absorbance at 405 nm (ε mM = 10.5), which is proportional to the enzyme activity.
Renal histological examination. The harvested right kidneys were kept in 10% formal saline and then embedded in paraffin blocks. Transverse sections of 5 μm thickness were prepared and stained with Haematoxylin and Eosin (H&E) and inspected blindly by a pathologist under a light microscope (BX43, Olympus, Tokyo, Japan) and photographed using the Cellsens dimension software connected to the Olympus DP27 camera. In five non-overlapping microscopic fields per animal (n = 3 each), the collective renal histopathological damage was assessed as the sum of individual lesions representing congestion of glomerular tufts/renal blood vessels (CO), inflammatory cell infiltration (IF), necrosis of tubular epithelium (NC), and renal cast (RC) that were graded/ scored from 0 to 5 according to the involved percent of the kidney: 0 = normal; 1 = mild (< 10%); 2 = moderate (10-25%); 3 = severe (25-50%); 4 = very severe (50-75%); 5 = extensive damage (> 75%) 41 .
Statistical analysis. Parametric data were expressed as means ± SD (n = 6 each). Comparisons between means were carried out using a one-way analysis of variance (ANOVA) followed by the Tukey's Multiple Comparison post hoc test. Non-parametric data are presented as median with range and analyzed using Kruskal Wallis test followed by the post hoc Dunn's test. A probability level of less than 0.05 was accepted as being significant in the used statistical tests. www.nature.com/scientificreports/ succeeded to bring both serum creatinine and BUN to the normal level and markedly abridged cystatin C by 82% and KIM-1 by 78%, as compared to the insult.

AEA improves renal function in
AEA turns off the IP3/NFATc1 signaling pathway in HgCl 2 -induced AKI. On the molecular level, Fig. 2 shows that HgCl 2 has markedly boosted the protein contents of (A) IP3 and (B) NFATc1 to 13 and 14 folds, respectively, compared to the control group. Treatment with AEA, on the other hand, noticeably quelled the contents of IP3 and NFATc1 by 84 and 79%, respectively, compared to HgCl 2 group.

AEA corrects the redox imbalance and apoptotic cell death mediated by HgCl 2 -induced AKI.
Apart from its inflammatory effect, HgCl 2 increased also oxidative stress ( Fig. 5) signified by the 20-fold increase in (A) MDA content and the depletion of the non-enzymatic defense molecule (B) GSH to 5% compared to the control value. These effects were associated with an apoptotic cascade ( Fig. 6), where HgCl 2 depleted (A) WNT-5A to reach 18% only and decreased the anti-apoptotic marker (B) BCL-2 by 88% but heightened the apoptotic enzyme (C) caspase-3 (12 folds) as compared to the normal group. On the other hand, AEA antioxidant effect was validated by the significant decrease in MDA and the increased GSH renal contents, as compared to the insult-related values. Furthermore, the AEA anti-apoptotic character was reflected by the decrease in caspase-3 by 77% but the upsurge in BCL-2 content and the 5-fold elevation in WNT-5A compared to the HgCl 2 values. www.nature.com/scientificreports/ AEA corrects the histopathological alterations induced by HgCl 2 in the rat kidney. Figure 7 demonstrates the kidney photomicrograph sections (H&E; scale bar 25 and 50 µm). The sections of (B and C) HgCl 2 untreated group show notable histopathological damage characterized by congestion of glomerular tufts, marked coagulative necrosis of epithelial lining renal tubules, renal cast in the lumen of renal tubules, and inflammatory cells infiltration compared to the section of (A) the control group, which depicts the normal architecture of renal parenchyma. However, an improved picture is spotted in section of (D) the AEA-treated group showing less epithelial coagulative necrosis of renal tubules compared to the insult. Panel E represents their effect on the collective scores of the lesions, whereas the individual lesion scores are presented in Table 1.

Discussion
The current study is the first to address the renoprotective effect of AEA against HgCl 2 -induced AKI evidenced by the improved early (cystatin C/ΚIM-1/IL-18) and late (creatinine/BUN) kidney injury diagnostic markers. The ability of AEA to suppress the IP3/NFATc1 axis offers one mechanism against the HgCl 2 -induced cytotoxicity. Besides, the anti-inflammatory potential of AEA was evidenced by the inhibition of the HMGB-1/RAGE hub and its downstream NF-κB p65, which was associated with a decrease in the caspase-1/IL-18 cue, despite the unpredicted decrease of the transcription factor PPAR-γ that was sharply boosted by HgCl 2 . Additionally, AEA pinned down its antioxidant capacity by suppressing lipid peroxidation and augmenting the defense molecule GSH. By hampering the heavy metal-mediated decrease of WNT-5A/BCL-2 and increase in caspase-3, AEA promoted cell survival and reduced apoptosis in the kidney. Our results showed that HgCl 2 boosted the IP3/NFATc1 axis, whereas treatment with AEA has abated it. HgCl 2 -induced AKI is known to be mediated partly by disturbing Ca 2+ homeostasis to increase its intracellular concentration and hence renal cytotoxicity, an effect that was mediated by ER injury 5 , mitochondrial dysfunction, and activation of PLC signal 6 . The current results offer a new mechanism by stimulating the IP3/NFATc1. Indeed, both IP3 and Hg have been shown to stimulate Ca 2+ release from endogenous stores by activating the IP3R, which in turn activates the Ca 2+ -dependent phosphatase calcineurin 42,43 . The latter dephosphorylates/ activates NFATc1 resulting in its nuclear accumulation and transcriptional activity 42,44 . Of note, previous studies have tethered activated NFATc1 to kidney injury, leading to proteinuria, glomerulosclerosis, and I/R-induced AKI [45][46][47] , verities that brace the present injurious role of IP3/NFATc1 in the HgCl 2 -induced AKI. A previous clinical study also showed that the use of cyclosporine A, an inhibitor of calcineurin, ameliorated idiopathic Figure 2. Effect of AEA on renal contents of (A) IP3 and (B) NFATc1 in HgCl 2 -induced AKI in rats. Data are expressed as the mean of 6 rats ± SD. *P < 0.05 and ***P < 0.001 compared with CONT and @@@ P < 0.001 compared with HgCl 2 groups using one-way ANOVA followed by Tukey's Multiple Comparison post hoc test. AEA anandamide, AKI acute kidney injury, CONT control, HgCl 2 mercuric chloride, IP3 inositol trisphosphate, NFATc1 nuclear factor of activated T-cells cytoplasmic-1. www.nature.com/scientificreports/ membranous nephropathy 48 , as well as proteinuria 49 . Thus, the inhibition of IP3/NFATc1 in the AEA-treated group offers one mechanism for its renoprotection, which may be attributed to a decrease in Ca 2+ , a notion that can be clarified by an earlier study 50 using activated murine bone marrow-derived mast cells. These co-authors provided evidence for the important role of the AEA-induced CB2R/GPR55 heterodimer in limiting the rise of intracellular Ca 2+ to suppress mast cell degranulation that was not CB1R dependent. The current model depleted WNT-5A to be markedly increased by the AEA administration. WNT-5A is an upstream protein of multiple signaling pathways including the non-canonical WNT cascade to increase intracellular Ca 2+ partly by stimulating the IP3/NFATc1 axis to trigger cytotoxicity 8,18 . However, as proven herein, its beneficial effect is not related to the IP3/NFATc1 cue but may rely on its anti-inflammatory capacity, a notion that concurs with earlier studies pointing to its anti-inflammatory capacity 19,20,51 .

Scientific Reports
Additionally, activated WNT-5A can play a role in reducing cell demise by regulating cell survival and resistance to apoptosis in different cell lines as previously documented 52,53 . The current results showed that the AEAtreated group displayed anti-apoptotic and cell survival potentials evidenced by increasing the anti-apoptotic molecule BCL-2 and inhibiting caspase-3, besides augmenting WNT-5A, which was reported to also mediate the formation of BCL-2 54 . The reduction of cystatin C and KIM-1 is another culprit for AEA-mediated cell survival, where cystatin C has been reported to downregulate the expression of BCL-2 55 and KIM-1, which was boosted after HgCl 2 injury here and hitherto 56 was expressed in all apoptotic tubules in a chronic cadmium nephrotoxicity model 57 facilitating apoptotic epithelial cell phagocytosis 58 .
The HgCl 2 -mediates injury is associated with the overproduction of reactive oxygen species (ROS) and the overwhelming of the defense system 3,4 . Thus, the antioxidant aptitude of AEA verified by curtailing the increased lipid peroxidation and elevating the renal content of the defense molecule GSH proffers a further mechanism for its ability to protect against HgCl 2 -induced AKI. The antioxidant effect of AEA may be partly linked to the suppressed IP3/NFATc1 axis, where the HgCl 2 -induced oxidative stress 59 was especially linked to hydrogen peroxide (H 2 O 2 ), which partakes in the generation of IP3 60 . Moreover, it was reported that the transcription factor PPAR-γ in HgCl 2 -induced AKI in rats. Data are expressed as the mean of 6 rats ± SD. **P < 0.01 and ***P < 0.001 compared with CONT and @@@ P < 0.001 compared with HgCl 2 groups using one-way ANOVA followed by Tukey's Multiple Comparison post hoc test. AEA anandamide, AKI acute kidney injury, CONT control, HgCl 2 mercuric chloride, HMGB-1 high mobility group box, pS536-NF-κB p65 nuclear factor-κB p65 phosphorylated at serine 536, PPAR-γ peroxisome proliferator-activated receptor gamma, RAGE receptor for advanced glycation end products. www.nature.com/scientificreports/  www.nature.com/scientificreports/ NFATc1, among others, participates in the transcriptional regulation of NADPH oxidase, one of the crucial oxidative enzymes 61 . Moreover, the restored WNT-5A can play a role in the antioxidant profile of AEA, where Lin et al. 62 recounted that exogenous administration of the antioxidant enzyme superoxide dismutase increased WNT-5A to protect against in a diabetic nephropathy model. Scarce data regarding the antioxidant effect of AEA are available; in one study AEA suppressed intracellular ROS and augmented GSH in H 2 O 2 -induced HT22 neuronal cells 63 . The latter co-workers owed the antioxidant response of AEA to the activation of CB1R, and recently, the selective CB2R agonists 1-phenylisatin and HU-308 have attenuated the cisplatin-induced renal oxidative stress 25,35 . Moreover, in an experimental myocardial infarction, Wang et al. 64 stated that stimulation of CB2R facilitates the antioxidant machinery to induce cellular antioxidant genes that control GSH synthesis and regeneration 65 . Moreover, Gallelli et al. 66 provided an overview of the role of CB and non-CB receptors for AEA in mediating its antioxidant effect not only CB2R.
In addition to the corrected redox balance, AEA confirmed its anti-inflammatory potential by inhibiting the inflammatory transcription factor NF-κB to support the results of a previous in vitro model 67 . A high degree of complexity characterizes the interaction between ROS and NF-κB, where, in a vicious cycle, the latter triggers the Figure 6. Effect of AEA on renal contents of (A) WNT-5A and (B) BCL-2, as well as (C) caspase-3 activity in HgCl 2 -induced AKI in rats. Data are expressed as the mean of 6 rats ± SD. **P < 0.01 and ***P < 0.001 compared with CONT and @@@ P < 0.001 compared with HgCl 2 groups using one-way ANOVA followed by Tukey's Multiple Comparison post hoc test. AEA anandamide, AKI acute kidney injury, BCL-2 B-cell lymphoma-2, CONT control, HgCl 2 mercuric chloride. www.nature.com/scientificreports/ production of the superoxide anion radical responsible for the activation of the ROS/NF-κB cue 11 . Accordingly, by its antioxidant/anti-inflammatory properties, AEA interjects the ROS/NF-κB vicious cycle, affording further clarification of its ability to suppress IP3/NFATc1 signaling. Besides the induction of ROS, NF-κB is responsible for the expression of an array of pro-inflammatory mediators, including the renal injury marker IL-18 12 , a verity that is reflected herein and associated by inflammatory cell infiltration as shown in the microphotographs of HgCl 2 exposed kidney. Along with the boosted IL-18, HgCl 2 heightened its activator caspase-1 to suggest a possible role for inflammasome signaling in the HgCl 2 -induced AKI, a point that warrants investigation. Treatment with AEA, on the other hand, has sharply reduced both IL-18 and its activator, caspase-1, to match the findings of Li et al. 16 , who showed that AEA inhibited the NLRP3 inflammasome and its downstream axis caspase-1/IL-18. The endocannabinoid also reduced the renal inflammatory cell infiltration to corroborate the decline in this inflammatory cytokine.
The HMGB-1/RAGE hub was also bolstered by HgCl 2 to be markedly suppressed by AEA. When HMGB-1, an endogenous danger-associated molecular pattern molecule, is released from the nucleus in a ROS-dependent manner 68 , it reactivates NF-κB upon binding to its receptor RAGE in a hostile intercalating loop 69 . Hence, the aptitude of AEA to oppose the inflammatory effect of HgCl 2 and to inhibit the HMGB-1/RAGE cue, documented herein for the first time, pins down the anti-inflammatory capacity of AEA, besides its antioxidant effect. It is worth mentioning that the current antioxidant and earlier reported anti-TNF-α effects of AEA 70 may be responsible also for the anti-apoptotic effect of AEA since they can inhibit caspase-3 by both intrinsic and extrinsic death pathways, respectively.
The third transcription factor investigated herein is PPAR-γ, which was unexpectedly upregulated in the HgCl 2 group, a response that might be compensatory since the activation of PPAR-γ is well documented to antagonize NF-κB, ROS, and inflammation 71 . This assumption could be braced by earlier findings in spontaneously hypertensive rat aorta and mesenteric resistance arteries 72 . On the other hand, the AEA-associated decrease in PPAR-γ can be explained by the ability of the endocannabinoid to quench ROS and inhibit inflammation, as seen here, which no longer necessitates the upregulation of PPAR-γ.
To this end, we have reported the nephroprotective role of AEA, which amended HgCl 2 -induced AKI by multiple intersecting mechanisms. AEA reduced inflammation, oxidative stress, and apoptosis through the normal control group reveals the normal histological architecture of renal parenchyma (normal renal tubules and glomeruli). In contrast, the sections of the (B,C) HgCl 2 -untreated rats show notable histopathological damage characterized by congestion of glomerular tufts, marked coagulative necrosis of epithelial lining renal tubules, renal cast in the lumen of renal tubules, and inflammatory cells infiltration. On the other hand, the section of the (D) AEA-treated group shows an improved picture with milder congestion of glomerular tufts and renal blood vessels as well as renal cast in the lumen of some renal tubules. All black arrows point to the altered structure in the section (scale bar = 25;50). Panel (E) illustrates the collective kidney damage score scatter blot; and data were analyzed using the Kruskal-Wallis test followed by Dunn's post hoc test. Values were calculated from 5 fields of n = 3 rats/group; as compared with *P < 0.05 compared with CONT group. AEA anandamide, HgCl 2 mercuric chloride. www.nature.com/scientificreports/ activation of the WNT-5A/BCL-2 survival pathway and inhibiting the IP-3/NFATc1, HMGB-1/RAGE/NF-κB, and caspase-1/IL-18 cues.

Data availability
All data generated or analyzed during this study are included in this article.
Received: 28 November 2022; Accepted: 12 July 2023 Table 1. Effect of AEA on individual kidney damage scores induced by HgCl 2 in rats. Data are presented as median (min-max) and analyzed using the Kruskal-Wallis test followed by Dunn's post hoc test. Values were calculated from 5 fields of n = 3 rats/group. Relative to the CONT (*P < 0.05) and HgCl 2 ( @ P < 0.05) groups. AEA anandamide, CO congestion of glomerular tufts and renal blood vessels, HgCl 2 mercuric chloride, IF inflammatory cells infiltration, NC necrosis of tubular epithelium, RC renal cast.